Oxidative stress has a large imprint upon biology: It determines the structure of microbial communities, is central to the action of the innate immune system, and is suspected of underlying a variety of human pathologies. Several basic questions frame the field: How are oxidants formed, and in what quantities? What are the specific biomolecules that they damage most rapidly? How do cells defend themselves? Why does oxidant sensitivity differ among organisms? Mechanistic details have primarily emerged from studies of the model bacterium E. coli. This application proposes to deepen and broaden the understanding. To date the reactive oxygen species (ROS) O2 and H2O2 are known to disrupt growth primarily by inactivating cluster-dependent dehydratases and non- - redox mononuclear iron enzymes. Dehydratases are vulnerable in virtually all organisms. However, it appears that the mononuclear enzymes of some aerobes acquired resistance by employing divalent metals other than iron. Such an evolutionary adaptation would be fascinating, as it would recapitulate a tactic that E. coli invokes during oxidative stress. Aim One will test whether this substitution is driven by changes in the intracellular metal pools or by modifications of the enzymes, and it will probe whether the shift in metallation exerts a cost in catalytic efficiency. Aim Two investigates the hypothesis that radical SAM enzymes comprise a novel third class of ROS- sensitive enzyme. Circumstantial data support the idea: they employ over-oxidizable peripheral iron-sulfur clusters, and E. coli responds to stress by replacing one of these enzymes with a cluster-free analogue. Yet those clusters might plausibly be protected in vivo either by bound SAM or by rapid re-reduction via their native electron donor. If this enzyme family is ROS-sensitive, then stress will affect a broad range of cell processes. Transcriptomic analyses have revealed surprising strategies by which E. coli copes with ROS, and Aim Three probes two newly discovered ones. First, the ClpSA and ClpX unfoldases somehow stabilize branched- chain biosynthesis during periods of H2O2 stress. The mechanism may be that by acting as chaperones the Clp proteins protect apo-dehydratases from aggregation and/or proteolysis. Thus damaged clusters can be re- built. Second, the data reveal that H2O2-stressed cells induce exonuclease III, a key enzyme that is specifically required to repair oxidative DNA lesions. The work will determine how its expression is controlled, whether other repair enzymes are co-regulated, and which enzymes are needed to allow growth in the face of environmental H2O2. Finally, Aim Four extends prior investigations of the molecular basis of obligate anaerobiosis. Previous work identified two key enzymes that are poisoned upon aeration of a model anaerobe; the next step is to resolve the underlying mechanisms. As proof of principle, this Aim will culminate in an effort to construct a derivative strain whose central metabolism is not blocked by oxygen. These projects are selected to fill in key pieces of the puzzle of oxidative stress. They are closely interconnected, with the mechanistic theme being the incompatibility of oxygen and iron-centered metabolism. Each Aim derives directly from data obtained during the current funding period.